1. Field:
This disclosure is concerned generally with the preparation, purification and use of hyaluronic acid and its salts and specifically with the preparation of hyaluronic acid from a microbiological source.
2. Prior Art:
Hyaluronic acid is a naturally occurring high molecular weight polysaccharide having an empirical formula of (C.sub.14 H.sub.20 N Na O.sub.11)n where n&gt;1000. The general structure of hyaluronic acid is illustrated in Merck Index, Ninth Ed. (3rd printing, 1978), at page 624. It is well known that hyaluronic acid and its salts, hereafter collectively referred to as HA, can be obtained from at least three sources: human umbilical cords, rooster combs and certain bacterial cultures such as group A and C hemolytic streptococci. To the best of our knowledge, however, only umbilical cords and rooster combs are used as sources for commercially available HA. This is somewhat surprising in view of certain disadvantages associated with using those two sources (e.g. relatively low yields, contamination with chondroitin sulfate, and labor intensive processing and purification steps).
Since HA is found in aqueous and vitreous humor and the synovial fluid of mammalian joints, there has been considerable interest in obtaining purified HA for use as a fluid replacement to correct pathological conditions in the eye and in joints. The preparation of HA from rooster combs and human umbilical cords and its use in eye and joint applications is described in U.S. Pat. No. 4,141,973 to E. A. Balazs. That patent also provides a detailed review of the technical literature describing the isolation, characterization and uses of HA.
U.S. Pat. No. 4,303,676, also to E. A. Balazs, describes cosmetic formulations containing sodium hyaluronate fractions in various molecular weight ranges made from rooster combs. U.S. Pat. No. 4,328,803 to L. G. Pape discloses the use of an ultrapure hyaluronic acid salt in eye surgery. The HA product used was a sodium hyaluronate salt available under the registered trademark HYARTIL.RTM. from Pharmacia, Inc. and obtained in commercial quantities from rooster combs.
The only literature found which describes extraction of hyaluronic acid from bacteria (see Kjem and Lebech, Acta Path. Microbiol. Scand. Sect. B, 84:162-164, 1976) uses a media and process which are unacceptable for some purposes. The described media will not support growth of most Streptococci. The described process begins with heat killing the Streptococci. This extracts the organisms, releasing numerous internal contaminants which are more likely to be reactive and which are difficult to remove from the final product. Therefore, it is likely the resulting HA could not be used for injection into mammals.
Because the medical applications of HA require that the HA be injected into a mammalian body (e.g. as a fluid replacement), it is very important that the injected products be as pure as possible to avoid reactivity problems. This importance of purity is described in U.S. Pat. No. 4,141,973 which describes an ultrapure HA product prepared from rooster combs or, alternatively, from human umbilical cords. In addition to purity, it appears that control of molecular weight of an HA product is very important (e.g. the 4,141,973 patent suggests an average molecular weight of at least 750,000 and U.S. Pat. No. 4,303,676 suggests having two distinct fractions of controlled molecular weight, one low and one high). Although there is a description of a high molecular weight (1,200,000) HA preparation of very high purity (i.e. less than 0.05% protein) in a paper by Swann, Arch. Opthal. 88, pp. 544-8 (1972), we are unaware of any description of an HA product having the following advantages: (1) derivable from a microbiological source at relatively low costs, in high yields, and with low reactivity upon injection; (2) having a desirably high and closely controlled average molecular weight; and (3) being substantially free of protein and nucleic acid impurities. Quite surprisingly, we have found it is now possible to prepare such a product. Details of its preparation, characterization, and use are described below.